Varietal Thiols and Green Characters

The Australian Wine Research Institute

Compounds responsible for the The Australian Wine green character Research Institute

 Methoxypyrazines N OMe . IBMP, SBMP, IPMP N  Sulfur compounds 3-Isobutyl-2-methoxypyrazine . DMS, DES, DMDS (IBMP) . 2-Isobutylthiazole S  C6 compounds Dimethyl sulfide . (Z)-3-Hexen-1-ol (DMS) . (E)-2-Hexenal . (Z)-3-Hexenal OH . Hexanal . 1-Hexanol (Z)-3-Hexen-1-ol . Hexyl esters (cis-3-Hexen-1-ol) Grape sources of C6 flavours in wine The Australian Wine Research Institute

 cis-3-Hexen-1-ol precursors . Formed from unsaturated fatty acids after berry damage (usually upon crushing) . Derived from linolenic acid through enzyme cascade

LOX/HPL O ADH OH

Linolenic Acid (Z)-3-Hexenal (Z)-3-Hexen-1-ol

Lipoxygenase → hydroperoxide lyase → alcohol dehydrogenase The Australian Wine Modulating factors – cis-3-Hexen-1-ol Research Institute

 Viticulture OH (Z)-3-Hexen-1-ol . Enzymatic formation via LOX pathway leads to C6 compounds (cis-3-Hexen-1-ol)

. Differs between varieties and during berry development (e.g. Riesling vs Cabernet Sauvignon)

. Highest at pre-veraison in line with unsaturated fatty acid levels – decline in linolenic acid with ripening

. Higher in skin (from press cake) than must at all ripening stages The Australian Wine Modulating factors – cis-3-Hexen-1-ol Research Institute

 Winemaking (Z)-3-Hexen-1-ol (cis-3-Hexen-1-ol) . Time and temperature of skin contact – similar extraction from 15-28 oC with max after 10-15 h, continual increase during contact time at 10 oC after 25 h

. Relatively stable but SO2 and enzymatic activity have effects – O2 needed for formation . Esterification to the acetate – from green (alcohol) to green/floral/fruity (ester)  Storage . Not affected by storage in presence of oxygen . Minimal change with storage on lees for up to seven months . Unaffected by short-term oxidative storage in presence of phenolics . Slow decline with storage for 210 days but no impact from different

SO2 levels

The Australian Wine Summary Research Institute

 Green flavours in wine are caused by a number of different compound classes, with vastly different potencies

 Compound origins are in the grape, often in precursor form

 Viticultural practices and harvesting decisions can impact on green flavours

 Green flavours may be desirable, adding complexity or typicity to wine styles

# 4

The Australian Wine Sensory impact of cis-3-Hexen-1-ol Research Institute

 cis-3-Hexen-1-ol – cut grass, herbaceous, leafy; 400 μg/L (Z)-3-Hexen-1-ol threshold (cis-3-Hexen-1-ol)

 Typically not found above threshold in most studies

 Found in wine up to . 650 µg/L in young red wines (highest in Tempranillo) . 800 µg/L in aged red wines . 75 µg/L in Gewurztraminer # 4 . 600 µg/L in some Italian and Spanish white wine varieties (Falanghina and Macabeo)

The Australian Wine Varietal thiols – impact odorants Research Institute

 Polyfunctional thiols are especially potent and have some of the lowest aroma thresholds of any food odorant  Varietal thiols are important impact odorants in some wines e.g. Sauvignon Blanc Thiol Perception Aroma OAV threshold

4-MMP 3 ng/L blackcurrant Up to 30 box tree passionfruit

3-MH 60 ng/L grapefruit Up to 210 passionfruit

passion-fruit 3-MHA 4 ng/L Up to 195 box tree

sweaty

Darriet et al. Flavour Fragr. 1995, 10, 385-392 Tominaga et al. Vitis 1996, 35, 207-210 8 Tominaga et al. Flavour Fragr. 1998, 13, 159-162 The Australian Wine Volatile thiol sensory descriptors Research Institute

 Individual volatile thiols contribute tropical aromas to wine, 3MH also citrus aroma  Volatile thiol combinations had aromas of tropical & cooked green vegetal at both levels, and at high levels also cat urine/sweaty  4MMP does not contribute any distinctive sensory properties at high levels  At high concentrations 3MHA is responsible for cat urine/sweaty aromas The Australian Wine Consumer preference of volatile thiols Research Institute

There was an optimal level of cat urine/sweaty attribute for one group of consumers identified

The majority of consumers preferred the samples with ‘green’ attributes, with a minority strongly preferring the ‘fruit’ and ‘estery’ flavours

Clear linking of volatile thiols in Sauvignon Blanc wines, their associated sensory attributes and effects on consumer preference Grape varieties containing volatile thiol The Australian Wine compounds Research Institute

White varieties Red varieties

Sauvignon Blanc Petit Manseng Cabernet Franc

Chardonnay Pinot Blanc Cabernet Sauvignon Chenin Blanc Pinot Gris Grenache Colombard Riesling Merlot Gewürztraminer Scheurebe Pinot Noir Gros Manseng Semillon Koshu Sylvaner Maccabeo Tokay Muscat Muscadet Petit Arvine Volatile thiol concentrations in wines The Australian Wine from around the world Research Institute

500 14000 450 4MMP 3MH 12000 400 350 10000

300 8000 250 6000 200 150 4000

100 concentration 3MH (ng/L) 4MMP concentration4MMP (ng/L) 2000 50 0 0 South Africa Australia France New Zealand South Africa Australia France New Zealand (n=12) (n=154) (n=141) (n=136) (n=12) (n=154) (n=121) (n=61) Country of origin Country of origin

4000

3500 3MHA 3000 2500 2000 1500 1000

3MHA concenrtation 3MHA (ng/L) 500 0 South Africa Australia France New Zealand (n=12) (n=154) (n=40) (n=136) Country of origin The Australian Wine Varietal thiol formation Research Institute

 Optimise formation and maximise stability of varietal thiols  Need to further understand precursor formation (Stress response : Kobayashi et al)  Yeast plays a key role in thiol release into wine  Need to understand relationship between precursors and free thiols

NH 2 H O HO N OH N O O H O S

OH Glut-3-MH

O H 2N O H yeast SH yeast S H O S O H O O H CSL 3 -M H ATF 3 -M H A Other Cys-3-MH Intermediates 13 from grapes winemaking The Australian Wine Modulation of volatile thiol precursors Research Institute

3MH precursor

Methoxypyrazines 4MMP precursor

• 3MH precursors are mainly found in the skins of grape berries • 4MMP precursors are mainly found in the flesh of grape berries The Australian Wine HPLC-MS/MS analysis of precursors Research Institute

 Amount of precursors measured in SAB juice:

Cys-3-MH 21 – 55 µg/L

Glut-3-MH 245 – 696 µg/L

 Also found precursors in other varieties (in the juice) generally:

Sauvignon Blanc > Pinot Gris > Chardonnay > Riesling

Capone et al. JAFC 2010, 58, 1390-1395 15

Precursor Grape studies The Australian Wine Research Institute

 5 different SAB clones in the same location in Adelaide Hills of South Australia

. Ripening

. Transportation / Holding

16 Concentration of 3MH during ripening: The Australian Wine Clone effects Research Institute

450

400 Q9720

/L) H5V10 ng

( 350 F7V7

3MH 3MH 300 5385 250 FV46

200

150 Concentration Concentration of

100

0 Veraison Mid Ripening Pre-harvest Harvest After Ferm

17 Capone et al. 2011, JAFC. 59: 4649-4658 Amount of 3-MH precursors during ripening The Australian Wine Research Institute

450

400 (R)-Glut 3-MH

350 (S)-Glut 3-MH

300 (R)-Cys 3-MH

250 (S)-Cys 3-MH

200

150 Concentration (µg/L) Concentration 100

Clone 1 Clone Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 2 Clone 3 Clone 4 Clone 5 Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone Veraison Mid Ripening Pre-harvest Harvest 18 Capone et al. 2011, JAFC. 59: 4649-4658 Effect of transportation on precursor The Australian Wine concentration Research Institute

No SO2 No SO2 50 SO2 50 SO2 50 SO2 500 SO2 500 SO2

No Asc 100 Asc No Asc 100 Asc 500 Asc No Asc 500 Asc

Analysed shortly after machine harvesting then ……..

19 The Australian Wine Research Institute

794 km 794 km

20 The Australian Wine Research Institute

794 km 794 km

21 The Australian Wine Effect of transportation on precursors Research Institute

1400

Cys-3-MH 1200 Glut-3-MH 1000

800

600

400

200

Precursor Concentration PrecursorConcentration (nmol/L)

50SO2_noasc 50SO2_noasc

noSO2_noasc noSO2_noasc

50SO2_100asc 50SO2_500asc 50SO2_100asc 50SO2_500asc

noSO2_100asc 500SO2_noasc noSO2_100asc 500SO2_noasc

500SO2_500asc 500SO2_500asc Prior to Transportation After Transportation 22 Capone et al. 2011, JAFC. 59: 4659-4667 The Australian Wine Modulation of volatile thiol precursors Research Institute

• Glutathione 3MH precursor is more abundant than Cysteine 3MH precursor, regardless of grape variety • 3MH precursors are affected by ripening. • 4MMP precursor peaked early in ripening season, at approx. 10°Beame • Mild water stress & moderate Nitrogen supply increased volatile thiols in wine • Foliage Copper spray pre-veraison decreased volatile thiols in wine • Foliar Nitrogen fertiliser with & without Sulfur increased volatile thiols in wine • Botrytis infection affects the levels of volatile thiols in the wine • 4MMP precursor found in free run juice & light pressings • 3MH precursor extracted mainly during skin contact – particularly longer periods of maceration and higher temperatures (18-20°C) Formation pathway to 3-MH The Australian Wine Research Institute

24 Conclusions – Factors affecting precursor The Australian Wine concentration in fruit Research Institute

 Ripening - Low levels of precursors until commercial harvest

 Transportation – inc. precursor for Cys and Glut

SO2 and Ascorbic acid – a combination of both optimum – very high SO2 suppresses conjugate formation

 Glut-3-MHAl – tentatively identified as intermediate between (hexenal + glutathione) and Glut-3-MH for the first time

 Cysgly-3-MH – Confirmed presence, is short lived

The Australian Wine Modulation of volatile thiols Research Institute

• Yeast selection • Higher fermentation temperatures increased volatile thiol levels (20°C compared to 13°C) • 3MH decreased during malolactic fermentation and barrel ageing • The addition of Sulfur dioxide stabilised 3MH and 4MMP levels in wine • Cork closures decreased the levels of 3MH and 3MHA in wine • 3MHA levels decreased dramatically within the first year of bottling • Addition of Copper as a wine fining agent decreased volatile thiol levels • In-mouth release of volatile thiol precursors by saliva bacteria

Yeast strains can release The Australian Wine differing levels of volatile thiols Research Institute

VL3 4MMP NT116 QA23 X5 Estery** L2056 5.0 VIN7 VIN13 Stale beer/ 4.0 Floral** 3MH yeasty** 3.0 2.0

1.0

0.0 Passion- Sweaty** fruit** 3MHA

Capsicum* Box * hedge**

Modified from Swiegers et al. (2009) The Australian Wine Modulation of volatile thiols Research Institute

The Australian Wine Flavour optimisation – the future Research Institute

 Be able to predict concentrations of volatiles from:

29 The Australian Wine Acknowledgements Research Institute

AWRI UA Dimitra Capone David Jeffery Kevin Pardon Mark Sefton Toni Cordente Yoji Hayasaka Industry Partners Ellie King Casella Winery Katryna van Leeuwen Steve Warne Cory Black Frank Mallamace

Australia’s grapegrowers and winemakers through their investment body, the Grape and Wine Research Development Corporation, with matching funds from the Australian government 30

The Australian Wine Sensory impact of 3-MHA Research Institute

 3-mercaptohexyl acetate - passionfruit, box tree, sweaty

 4 ng/L threshold

 Found in Aust. wine up to 3,000 ng/L

 Found in NZ wine up to 12,000 ng/L # 3  Final concentration in your glass - 740 ng/L

The Australian Wine Sensory impact of 3-MH Research Institute

 3-mercaptohexen-1-ol - grapefruit, passionfruit, leafy

 60 ng/L threshold

 Found in wine up to 210 ng/L

 Final concentration in your glass - 7040 ng/L # 5

Sensory impact of thiol mix The Australian Wine (3-MHA + 4-MMP + 3-MH) Research Institute

 Individual aroma characteristics

- grapefruit, passionfruit, leafy, box tree, sweaty

 Combined aroma characteristics - cooked green veg, tropical

Spiked levels in your wine:  3MHA - 740 ng/L # 6

 3MH – 7040 ng/L

 4MMP – 40 ng/L